Uses of synthetic peptides corresponding to telopeptide sequences of cross-linked type I collagen metabolites

ABSTRACT

Peptides synthesized to match the human α1 (I) and α2 (I) amino-telopeptide sequences of type I collagen degradation products in body fluids, preferably either Asp-GIu-Lys-Ser-Thr-Gly-Gly (SEQ ID NO:5) or Gln-Tyr-Asp-Gly-Lys-Gly-Val-Gly (SEQ ID NO:6). used as calibrators and antigens in immunoassays for detecting type I collagen degradation products in body fluids.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 09/662,751,filed Sep. 15, 2000 now abandoned, which is a continuation ofapplication Ser. No. 09/457,440, filed Dec. 7, 1999 (U.S. Pat. No.6,143,511), which is a continuation of application Ser. No. 09/347,503,filed Jul. 2, 1999 (U.S. Pat. No. 6,100,379), which is a continuation ofapplication Ser. No. 09/237,176, filed Jan. 25, 1999 (U.S. Pat. No.6,048,705), which is a continuation of application Ser. No. 09/047,144,filed Mar. 24, 1998 (U.S. Pat. No. 5,962,639), which is a continuationof application Ser. No. 08/803,960, filed Feb. 21, 1997 (abandoned),which is a continuation of application Ser. No. 08/664,102, filed Jun.13, 1996 (U.S. Pat. No. 5,677,198), which is a continuation ofapplication Ser. No. 08/567,618, filed Dec. 4, 1995 (U.S. Pat. No.5,656,439), which is a continuation of application Ser. No. 08/457,831,filed Jun. 1, 1995 (U.S. Pat. No. 5,576,189), which is a continuation ofapplication Ser. No. 08/221,705, filed Apr. 1, 1994 (U.S. Pat. No.5,473,052), which is a continuation of application Ser. No. 07/614,719,filed Nov. 21, 1990 (U.S. Pat. No. 5,300,434), which is acontinuation-in-part of U.S. application Ser. No. 07/448,881, filed Dec.1, 1989 (U.S. Pat. No. 5,140,103), which is a continuation-in-pan ofU.S. application Ser. No. 07/118,234, filed Nov. 6, 1987 (U.S. Pat. No.4,973,666).

This invention was made with government support under grants AR37318 andAR36794 awarded by the National Institutes of Health. The government hascertain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to methods for detecting and monitoringcollagen degradation in vivo. More specifically, it relates to methodsfor quantitating cross-linked telopeptides produced in vivo upondegradation of collagen types II and III.

BACKGROUND OF THE INVENTION

Three known classes of collagens have been described to date. The ClassI collagens, subdivided into types I, II, III, V, and XI, are known toform fibrils. These collagens are all synthesized as procollagenmolecules, made up of N-terminal and C-terminal propeptides, which areattached to the core collagen molecule. After removal of thepropeptides, which occurs naturally in vivo during collagen synthesis,the remaining core of the collagen molecule consists largely of atriple-helical domain having terminal telopeptide sequences which arenontriple-helical. These telopeptide sequences have an importantfunction as sites of intermolecular cross-linking of collagen fibrilsextracellularly.

The present invention relates to methods of detecting collagendegradation based on assaying for particular cross-linked telopeptidesproduced in vivo upon collagen degradation. In the past, assays havebeen developed for monitoring degradation of collagen in vivo bymeasuring various biochemical markers, some of which have beendegradation products of collagen. For example, bone turnover associatedwith Paget's disease has been monitored by measuring small peptidescontaining hydroxyproline, which are excreted in the urine followingdegradation of bone collagen. Russell et al., Metab. Bone Dis. and Rel.Res. 4 and 5, 255-262 (1981); and Singer, F. R., et al., Metabolic BoneDisease, Vol. II (eds. Avioli, L. V. and Kane, S. M.), 489-575 (1978),Academic Press, New York.

Other researchers have measured the cross-linking compound pyridinolinein urine as an index of collagen degradation in joint disease. See, forbackground and for example, Wu and Eyre, Biochemistry, 23:1850 (1984);Black et al., Annals of the Rheumatic Diseases, 48:641-644 (1989);Robins et al.; Annals of the Rheumatic Diseases, 45:969-973 (1986); andSeibel et al., The Journal of Rheumatology, 16:964 (1989). In contrastto the present invention, some prior researchers have hydrolyzedpeptides from body fluids and then looked for the presence of individualhydroxypyridinium residues. None of these researchers has reportedmeasuring a telopeptide containing a cross-link that is naturallyproduced in vivo upon collagen degradation, as in the present invention.

U.K. Patent application GB 2,205,643 reports that the degradation oftype III collagen in the body is quantitatively determined by measuringthe concentration of an N-terminal telopeptide from type III collagen ina body fluid. In this reference, it is reported that cross-linkedtelopeptide regions are not desirable. In fact, this reference reportsthat it is necessary to use a non-cross-linked source of collagen toobtain the telopeptide. The peptides of the present invention are allcross-linked. Collagen cross-links are discussed in greater detailbelow, under the heading “Collagen Cross-Linking.”

There are a number of reports indicating that collagen degradation canbe measured by quantitating certain procollagen peptides. The presentinvention involves telopeptides rather than propeptides, the two beingdistinguished by their location in the collagen molecule and the timingof their cleavage in vivo. See U.S. Pat. No. 4,504,587; U.S. Pat. No.4,312,853; Pierard et al., Analytical Biochemistry 141:127-136 (1984);Niemela, Clin. Chem., 31/8:1301-1304 (1985); and Rohde et al., EuropeanJournal of Clinical Investigation, 9:451-459 (1979).

U.S. Pat. No. 4,778,768 relates to a method of determining changesoccurring in articular cartilage involving quantifying proteoglycanmonomer or antigenic fragments thereof in a synovial fluid sample. Thispatent does not relate to detecting cross-linked telopeptides derivedfrom degraded collagen.

Dodge, J. Clin. Invest., 83:647-661 (1981) discloses methods foranalyzing type II collagen degradation utilizing a polyclonal antiserumthat specifically reacts with unwound alpha-chains and cyanagenbromide-derived peptides of human and bovine type II collagens. Thepeptides involved are not cross-linked telopeptides as in the presentinvention.

Amino acid sequences of human type III collagen, human proα1(II)collagen, and the entire preproα1(III) chain of human type III collagenand corresponding cDNA clones have been investigated and determined byseveral groups of researchers. See Loidl et al., Nucleic Acids Research12:9383-9394 (1984); Sangiorgi et al., Nucleic Acids Research,13:2207-2225 (1985); Baldwin et al., Biochem. J., 262:521-528 (1989);and Ala-Kokko et al., Biochem. J., 260:509-516 (1989). None of thesereferences specifies the structures of particular telopeptidedegradation products that could be measured to determine the amount ofdegraded fibrillar collagen in vivo.

In spite of the above-described background information, there remains aneed for effective and simple assays for determining collagendegradation in vivo. Such assays could be used to detect and monitordisease states in humans, such as osteoarthritis (type II collagendegradation), and various inflammatory disorders, such as vasculitissyndrome (type III collagen degradation).

Assays for type I collagen degradation, described in the parentapplication, U.S. Ser. No. 118,234, can be utilized to detect and assessbone resorption in vivo. Detection of bone resorption may be a factor ofinterest in monitoring and detecting diseases such as osteoporosis.Osteoporosis is the most common bone disease in man. Primaryosteoporosis, with increased susceptibility to fractures, results from aprogressive net loss of skeletal bone mass. It is estimated to affect15-20 million individuals in the United States. Its basis is anage-dependent imbalance in bone remodeling, i.e., in the rates ofsynthesis and degradation of bone tissue.

About 1.2 million osteoporosis-related fractures occur in the elderlyeach year including about 538,000 compression fractures of the spine,about 227,000 hip fractures and a substantial number of early fracturedperipheral bones. Twelve to 20% of the hip fractures are fatal becausethey cause severe trauma and bleeding, and half of the survivingpatients require nursing home care. Total costs fromosteoporosis-related injuries now amount to at least $7 billion annually(Barnes, O. M., Science, 236:914 (1987)).

Osteoporosis is most common in postmenopausal women who, on average,lose 15% of their bone mass in the 10 years after menopause. Thisdisease also occurs in men as they get older and in young amenorrheicwomen athletes. Despite the major, and growing, social and economicconsequences of osteoporosis, no method is available for measuring boneresorption rates in patients or normal subjects. A major difficulty inmonitoring the disease is the lack of a specific assay for measuringbone resorption rates.

Methods for assessing bone mass often rely on measuring whole-bodycalcium by neutron activation analysis or mineral mass in a given boneby photon absorption techniques. These measurements can give onlylong-term impressions of whether bone mass is decreasing. Measuringcalcium balances by comparing intake with output is tedious, unreliableand can only indirectly appraise whether bone mineral is being lost overthe long term. Other methods currently available for assessing decreasedbone mass and altered bone metabolism include quantitative scanningradiometry at selected bone locations (wrist, calcaneus, etc.) andhistomorphometry of iliac crest biopsies. The former provides a crudemeasure of the bone mineral content at a specific site in a single bone.Histomorphometry gives a semi-quantitative assessment of the balancebetween newly deposited bone seams and resorbing surfaces.

A urinary assay for the whole-body output of degraded bone in 24 hourswould be much more useful. Mineral studies (e.g., calcium balance)cannot do this reliably or easily. Since bone resorption involvesdegradation of the mineral and the organic matrix, a specificbiochemical marker for newly degraded bone products in body fluids wouldbe the ideal index. Several potential organic indices have been tested.For example, hydroxyproline, an amino acid largely restricted tocollagen, and the principal structural protein in bone and all otherconnective tissues, is excreted in urine. Its excretion rate is known tobe increased in certain conditions, notably Paget's disease, a metabolicbone disorder in which bone turnover is greatly increased, as pointedout above. For this reason, urinary hydroxyproline has been usedextensively as an amino acid marker for collagen degradation. Singer, F.R., et al. (1978), cited hereinabove.

U.S. Pat. No. 3,600,132 discloses a process for determination ofhydroxyproline in body fluids such as serum, urine, lumbar fluid andother intercellular fluids in order to monitor deviations in collagenmetabolism. In particular, this inventor notes that in pathologicconditions such as Paget's disease, Marfan's syndrome, osteogenesisimperfecta, neoplastic growth in collagen tissues and in various formsof dwarfism, increased collagen anabolism or catabolism as measured byhydroxyproline content in biological fluids can be determined. Thisinventor measures hydroxyproline by oxidizing it to a pyrrole compoundwith hydrogen peroxide and N-chloro-p-toluenesulphonamide followed bycolorimetric determination in p-dimethyl-amino-benzaldehyde.

In the case of Paget's disease, the increased urinary hydroxyprolineprobably comes largely from bone degradation; hydroxyproline, however,generally cannot be used as a specific index. Much of the hydroxyprolinein urine may come from new collagen synthesis (considerable amounts ofthe newly made protein are degraded and excreted without ever becomingincorporated into tissue fabric), and from turnover of certain bloodproteins as well as other proteins that contain hydroxyproline.Furthermore, about 80% of the free hydroxyproline derived from proteindegradation is metabolized in the liver and never appears in the urine.Kiviriko, K. I. Int. Rev. Connect. Tissue Res. 5:93 (1970), and Weiss,P. H. and Klein, L., J. Clin. Invest. 48: 1 (1969).

Hydroxylysine and its glycoside derivatives, both peculiar tocollagenous proteins, have been considered to be more accurate thanhydroxyproline as markers of collagen degradation. However, for the samereasons described above for hydroxyproline, hydroxylysine and itsglycosides are probably equally non-specific markers of bone resorption.Krane, S. M. and Simon, L. S. Develop. Biochem., 22:185 (1981).

In addition to amino acids unique to collagen, various non-collagenousproteins of bone matrix such as osteocalcin, or their breakdownproducts, have formed the basis of immunoassays aimed at measuring bonemetabolism. Price, P. A. et al. J. Clin. Invest., 66: 878 (1980), andGundberg, C. M. et al., Meth. Enzymol., 107:516 (1984). However, it isnow clear that bone-derived non-collagenous proteins, though potentiallya useful index of bone metabolic activity are unlikely, on their own, toprovide quantitative measures of bone resorption. The concentration inserum of osteocalcin, for example, fluctuates quite widely both normallyand in metabolic bone disease. Its concentration is elevated in statesof high skeletal turnover but it is unclear whether this results fromincreased synthesis or degradation of bone. Krane, S. M., et al.,Develop. Biochem., 22:185 (1981), Price, P. A. et al., J. Clin. Invest.,66:878 (1980); and Gundberg, C. M. et al., Meth. Enzymol., 107:516(1984).

Collagen Cross-Linking

The polymers of most genetic types of vertebrate collagen require theformation of aldehyde-mediated cross-links for normal function. Collagenaldehydes are derived from a few specific lysine or hydroxylysineside-chains by the action of lysyl oxidase. Various di-, tri- andtetrafunctional cross-linking amino acids are formed by the spontaneousintra- and intermolecular reactions of these aldehydes within the newlyformed collagen polymers; the type of cross-linking residue variesspecifically with tissue type (see Eyre, D. R. et al., Ann. Rev.Biochem., 53:717-748 (1984)).

Two basic pathways of cross-linking can be differentiated for the banded(67 nm repeat) fibrillar collagens, one based on lysine aldehydes, theother on hydroxylysine aldehydes. The lysine aldehyde pathway dominatesin adult skin, cornea, sclera, and rat tail tendon and also frequentlyoccurs in other soft connective tissues. The hydroxylysine aldehydepathway dominates in bone, cartilage, ligament, most tendons and mostinternal connective tissues of the body, Eyre, D. R. et al. (1974) vidasupra. The operating pathway is governed by whether lysine residues arehydroxylated in the telopeptide sites where aldehyde residues will laterbe formed by lysyl oxidase (Barnes, M. J. et al., Biochem. J., 139:461(1974)).

The chemical structure(s) of the mature cross-linking amino acids on thelysine aldehyde pathway are unknown, but hydroxypyridinium residues havebeen identified as mature products on the hydroxylysine aldehyde route.On both pathways and in most tissues the intermediate,borohydride-reducible cross-linking residues disappear as the newlyformed collagen matures, suggesting that they are relatively short-livedintermediates (Bailey, A. J. et al., FEBS Lett., 16:86 (1971)).Exceptions are bone and dentin, where the reducible residues persist inappreciable concentration throughout life, in part apparently becausethe rapid mineralization of the newly made collagen fibrils inhibitsfurther spontaneous cross-linking interactions (Eyre, D. R., In: TheChemistry and Biology of Mineralized Connective Tissues, (Veis, A. ed.)pp. 51-55 (1981), Elsevier, New York, and Walters, C. et al., Calc.Tiss. Intl., 35:401-405 (1983)).

Two chemical forms of 3-hydroxypyridinium cross-link have beenidentified (Formula I and II). Both compounds are naturally fluorescent,with the same characteristic excitation and emission spectra (Fujimoto,D. et al. Biochem. Biophys. Res. Commun., 76:1124 (1977), and Eyre, D.R., Develop. Biochem., 22:50 1981)). These amino acids can be resolvedand assayed directly in tissue hydrolysates with good sensitivity usingreverse phase HPLC and fluorescence detection. Eyre, D. R. et al.,Analyte. Biochem., 137:380-388 (1984). It should be noted that thepresent invention involves quantitating particular peptides rather thanamino acids.

In growing animals, it has been reported that these mature cross-linksmay be concentrated more in an unmineralized fraction of bone collagenthan in the mineralized collagen (Banes, A. J., et al., Biochem.Biophys. Res. Commun., 113:1975 (1983). However, other studies on youngbovine or adult human bone do not support this concept, Eyre, D. R., In:The Chemistry and Biology of Mineralized Tissues (Butler, W. T. ed.) p.105 (1985), Ebsco Media Inc., Birmingham, Ala.

The presence of collagen hydroxypyridinium cross-links in human urinewas first reported by Gunja-Smith and Boucek (Gunja-Smith, Z. andBoucek, R. J., Biochem J., 197:759-762 (1981)) using lengthy isolationprocedures for peptides and conventional amino acid analysis. At thattime, they were aware only of the HP form of the cross-link. Robins(Robins, S. P., Biochem J., 207:617-620 (1982) has reported anenzyme-linked immunoassay to measure HP in urine, having raisedpolyclonal antibodies to the free amino acid conjugated to bovine serumalbumin. This assay is intended to provide an index for monitoringincreased joint destruction that occurs with arthritic diseases and isbased, according to Robins, on the finding that pyridinoline is muchmore prevalent in cartilage than in bone collagen.

In more recent work involving enzyme-linked immunoassay, Robins reportsthat lysyl pyridinoline is unreactive toward antiserum to pyridinolinecovalently linked to bovine serum albumin (Robins et al., Ann. Rheum.Diseases, 45:969-973 (1986)). Robins' urinary index for cartilagedestruction is based on the discovery that hydroxylysyl pyridinoline,derived primarily from cartilage, is found in urine at concentrationsproportional to the rate of joint cartilage resorption (i.e.,degradation). In principle, this index could be used to measure wholebody cartilage loss; however, no information on bone resorption would beavailable.

A need therefore exists for a method that allows the measurement ofwhole-body bone resorption rates in humans. The most useful such methodwould be one that could be applied to body fluids, especially urine. Themethod should be sensitive, i.e., quantifiable down to 1 picomole andrapidly measure 24-hour bone resorption rates so that the progress ofvarious therapies (e.g., estrogen) can be assessed.

SUMMARY OF THE INVENTION

The present invention is based on the discovery of the presence ofparticular cross-linked telopeptides in body fluids of patients andnormal human subjects. These telopeptides are produced in vivo duringcollagen degradation and remodeling. The term “telopeptides” is used ina broad sense herein to mean cross-linked peptides having sequences thatare associated with the telopeptide region of, e.g., type II and typeIII collagens and which may have cross-linked to them a residue orpeptide associated with the collagen triple-helical domain. Generally,the telopeptides disclosed herein will have fewer amino acid residuesthan the entire telopeptide domains of type II and type III collagens.Typically, the telopeptides of the present invention will comprise twopeptides linked by a pyridinium cross-link and further linked by apyridinium cross-link to a residue or peptide of the collagentriple-helical domain. Having disclosed the structures of thesetelopeptides herein, it will be appreciated by one of ordinary skill inthe art that they may also be produced other than in vivo, e.g.,synthetically. These peptides will generally be provided in purifiedform, e.g., substantially free of impurities, particularly otherpeptides.

The present invention also relates to methods for determining in vivodegradation of type II and type III collagens. The methods involvequantitating in a fluid the concentration of particular telopeptidesthat have a 3-hydroxypyridinium cross-link and that are derived fromcollagen degradation. The methods disclosed in the present invention areanalogous to those previously disclosed in U.S. Ser. No. 118,234, filedNov. 6, 1987, for determining the absolute rate of bone resorption invivo. Those methods involved quantitating in a body fluid theconcentration of telopeptides having a 3-hydroxypyridinium cross-linkderived from bone collagen resorption.

In a representative assay, the patient's body fluid is contacted with animmunological binding partner specific to a telopeptide having a3-hydroxypyridinium cross-link derived from type II or type IIIcollagen. The body fluid may be used as is or purified prior to thecontacting step. This purification step may be accomplished using anumber of standard procedures, including cartridge adsorption andelution, molecular sieve chromatography, dialysis, ion exchange, aluminachromatography, hydroxyapatite chromatography, and combinations thereof.

Other representative embodiments of quantitating the concentration ofpeptide fragments having a 3-hydroxypyridinium cross-link in a bodyfluid include electrochemical titration, natural fluorescencespectroscopy, and ultraviolet absorbance. Electrochemical titration maybe conducted directly upon a body fluid without further purification.However, when this is not possible due to excessive quantities ofcontaminating substances, the body fluid is first purified prior to theelectrochemical titration step. Suitable methods for purification priorto electrochemical detection include dialysis, ion exchangechromatography, alumina chromatography, molecular sieve chromatography,hydroxyapatite chromatography and ion exchange absorption and elution.

Fluorometric measurement of a body fluid containing a3-hydroxypyridinium cross-link is an alternative way of quantitatingcollagen degradation (and, hence, bone resorption, if type I peptidesare quantitated). The fluorometric assay can be conducted directly on abody fluid without further purification. However, for certain bodyfluids, particularly urine, it is preferred that purification of thebody fluid be conducted prior to the fluorometric assay. Thispurification step consists of dialyzing an aliquot of a body fluid suchas urine against an aqueous solution thereby producing partiallypurified peptide fragments retained within the nondiffusate (retentate).The nondiffusate is then lyophilized, dissolved in an ion pairingsolution and adsorbed onto an affinity chromatography column. Thechromatography column is washed with a volume of ion pairing solutionand, thereafter, the peptide fragments are eluted from the column withan eluting solution. These purified peptide fragments may then behydrolyzed and the hydrolysate resolved chromatographically.Chromatographic resolution may be conducted by either high-performanceliquid chromatography or microbore high performance liquidchromatography.

The invention includes peptides having structures identical to peptidesderived from collagen degradation, substantially free from other humanpeptides, which may be obtained from a body fluid. The peptides containat least one 3-hydroxypyridinium cross-link, in particular, a lysylpyridinoline cross-link or a hydroxylysyl pyridinoline cross-link, andare derived from the telopeptide region of type II or type III collagenlinked to one or more residues from a triple-helical domain, typicallyby the action of endogenous proteases and/or peptidases.

The structures of the type II and type III telopeptides are disclosedbelow. Information on the type I telopeptides, originally presented inU.S. Ser. No. 118,234, is also included.

Another aspect of the present invention involves assays for the peptidesdescribed herein in which the pyridinium rings are intact and cleaved.Since it is suspected that some cleavage of pyridinium rings occurs invivo, assays that detect both intact and cleaved pyridinium rings maylead to more accurate assessments of collagen degradation. In connectionwith this aspect of the present invention, specific binding partners tothe individual peptides containing intact or cleaved pyridinium rings,may be employed in the assays. Individual specific binding partners thatrecognize both types of peptides (both intact and cleaved pyridiniumring containing peptides) may be employed. Alternatively, specificbinding partners that discriminate between peptides containing theintact pyridinium ring and those in which the pyridinium ring iscleaved, could also be used.

Structure of Cross-Linked Telopeptides Derived from Type I Collagen

A specific telopeptide having a 3-hydroxypyridinium cross-link derivedfrom the N-terminal (amino-terminal) telopeptide domain of bone type Icollagen has the following amino acid sequence:

is hydroxylysyl pyridinoline or lysyl pyridinoline, and Gln is glutamineor pyrrolidine carboxylic acid.

The invention also encompasses a peptide containing at least one3-hydroxypyridinium cross-link derived from the C-terminal(carboxy-terminal) telopeptide domain of bone type I collagen. TheseC-terminal telopeptide sequences are cross-linked with either lysylpyridinoline or hydroxylysyl pyridinoline. An example of such a peptidesequence is represented by the formula:

is hydroxylysyl or lysyl pyridinoline.

Since the filing of U.S. Ser. No. 118,234, the inventor has discoveredevidence of two additional type I collagen telopeptides in body fluids,having the following structures:

These telopeptides may also be quantitated in body fluids in accordancewith the present invention.Structure of a Cross-Linked Telopeptide Derived from Type II Collagen

A specific telopeptide having a hydroxylysyl pyridinoline cross-linkderived from the C-terminal telopeptide domain of type II Collagen hasthe following amino acid sequence (referred to hereinbelow as the corepeptide structure):

wherein the cross-linking residue depicted as Hyl-Hyl-Hyl ishydroxylysyl pyridinoline (HP), a natural 3-hydroxypyridinium residuepresent in mature collagen fibrils of various tissues.

Amino-terminal telopeptides from type II collagen have not been detectedin body fluids, and it is suspected that potential peptides derived fromthe N-terminal telopeptide region of type II collagen are substantiallydegraded in vivo, perhaps all the way to the free HP cross-linking aminoacid.

Structure of Cross-Linked Telopeptides Derived from Type III Collagen

By analogy to the above disclosure, cross-linked peptides that arederived from proteolysis of human type III collagen may be present inbody fluids. These peptides have a core structure embodied in thefollowing parent structures:

is hydroxylysyl or lysyl pyridinoline, and Gln is glutamine orpyrrolidine carboxylic acid.

A likely cross-linked peptide derived from type III collagen in bodyfluids has the core structure:

that is derived from two α1(III)N-telopeptide domains linked to anhydroxylysyl pyridinoline residue (Hyl-Hyl-Hyl).

A second possible fragment of the C-telopeptide cross-linking domain,based on the collagen types I and II peptides observed in urine, has thecore structure:

Smaller and larger versions (differing by one to three amino acids oneach component chain) of these two peptides corresponding to the parentsequences shown above (FORMULAE VIII and IX) may also be present andmeasurable in body fluids. Analogous smaller and larger versions of eachof the peptides disclosed herein form part of the present invention aswell.

The invention generally includes all specific binding partners to thepeptides described herein. “Specific binding partners” are moleculesthat are capable of binding to the peptides of the present invention.Included within this term are immunological binding partners, such asantibodies (monoclonal and polyclonal), antigen-binding fragments ofantibodies (e.g., Fab and F(ab′)₂ fragments), single-chainantigen-binding molecules, and the like, whether made by hybridoma orrDNA technologies.

The invention includes fused cell hybrids (hybridomas) that producemonoclonal antibodies specific for the above-described collagen peptideshaving 3-hydroxypyridinium cross-links (both with an intact pyridiniumring and one that has been cleaved).

The invention further includes monoclonal antibodies produced by thefused cell hybrids, and those antibodies (as well as binding fragmentsthereof, e.g., Fab) coupled to a detectable marker. Examples ofdetectable markers include enzymes, chromophores, fluorophores,coenzymes, enzyme inhibitors, chemiluminescent materials, paramagneticmetals, spin labels, and radioisotopes. Such specific binding partnersmay alternatively be coupled to one member of a ligand-binding partnercomplex (e.g., avidin-biotin), in which case the detectable marker canbe supplied bound to the complementary member of the complex.

The invention also includes test kits useful for quantitating the amountof peptides having 3-hydroxypyridinium cross-links derived from collagendegradation in a body fluid. The kits may include a specific bindingpartner to a peptide derived from degraded collagen as disclosed herein.The specific binding partners of the test kits may be coupled to adetectable marker or a member of a ligand-binding partner complex, asdescribed above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a depiction of type II collagen and a proposal for the sourceof telopeptides. It is not established whether the two telopeptidesshown come from one collagen molecule as depicted in FIG. 1 or from twocollagen molecules.

FIG. 2 shows relative fluorescence (297 nm excitation; 390 nm emission)versus fraction number (4 ml), obtained during molecular sievechromatographic purification of cross-linked telopeptides. Cross-linkedtype II collagen telopeptides are contained in the fractions designatedII.

FIG. 3A shows relative fluorescence (330 nm excitation, 390 nm emission)versus elution time of fractions during ion exchange HPLC (DEAE-5PW).Cross-linked type II collagen telopeptides are contained in the fractiondesignated IV.

FIG. 3B shows absorbance (220 nm) versus elution time in minutes for thesame chromatogram.

FIG. 4A shows relative fluorescence (297 nm excitation, 390 nm emission)versus elution time of fractions during reverse phase HPLC. Cross-linkedtype II collagen telopeptides are eluted as indicated. The fractionsindicated by the bar (−) show evidence by sequence and compositionanalysis of the peptides indicated that retain or have lost the gly (G)and pro (P) residues.

FIG. 4B shows absorbance (220 nm) as a function of elution time duringreverse phase HPLC.

FIG. 5 compares the concentration of HP and LP in both cortical andcancellous human bone with age.

FIG. 6 depicts the cross-link molar ratios of HP to LP as a function ofage.

FIG. 7A shows relative fluorescence (297 nm excitation, >370 nmemission) as a function of elution volume during reverse phase HPLCseparation of cross-linked type I collagen N-telopeptides.

FIG. 7B shows relative fluorescence (297 nm excitation, >370 nmemission) versus elution volume during reverse phase HPLC separation ofcross-linked type I collagen C-telopeptides.

FIG. 8A shows relative fluorescence (297 nm excitation, >380 nmemission) as a function of elution time for the cross-linked type Icollagen telopeptides.

FIG. 8B shows relative fluorescence (297 nm excitation, >380 nmemission) as a function of elution time for the cross-linked type Icollagen telopeptides.

FIG. 9 shows results of binding experiments with the representativemonoclonal antibody HB 10611 and: the P1 peptide (Formula III herein,open squares); an α2 (I) N-telopeptideGln-Tyr-Asp-Gly-Lvs-Gly-Val-Gly-Cvs (SEQ ID NO:1), solid diamonds); andan α1 (I) N-telopeptide Tyr-Asp-Glu-Lys-Ser-Thr-Gly-Gly-Cys (SEQ IDNO:2), solid squares).

FIG. 10 shows a portion of the structure of the N-telopeptide region ofdecalcified human bone collagen. The P1 peptide (Formula III) isenclosed in a box; it contains an epitope that correlates with boneresorption.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Type II Collagen Telopeptides

The core peptide structure of the type II collagen peptides may be foundin body fluids as a component of larger peptides that bear additionalamino acids or amino acid sequences on one or more ends of the threepeptide sequences joined by the HP residue. FIG. 1 shows how type IIcollagen telopeptides, which are linked to a triple-helical sequence,may be produced in vivo from a human source using the proteolyticenzymes pepsin and trypsin. Smaller fragments that have lost amino acidsfrom the core peptide structure, particularly from the helical sequence,may also occur in body fluids. Generally, additions or deletions ofamino acids from the core peptide structure will involve from 1 to about3 amino acids. Additional amino acids will generally be determined bythe type II collagen telopeptide sequence that occurs naturally in vivo.As examples, peptides having the following structure:

can be isolated chromatographically from urine, and another ofstructure:

may also be isolated. In addition, glycosylated variants of the corestructure and its larger and smaller variants may occur in which agalactose residue or a glucosyl galactose residue are attached to theside chain hydroxyl group of the HP cross-linking residue. Each peak inthe graph shown in FIGS. 4A and 4B may correspond to a cross-linkedfragment of particular structure that may be quantitated for purposes ofthe present invention.

These structures are consistent with their site of origin in human typeII collagen fibrils at a molecular cross-linking site formed between twoα1(II) C-telopeptides and residue 87 of a triple-helical domain, theknown sequences about which are:

The isolated peptide fragments represent the products of proteolyticdegradation of type II collagen fibrils within the body. The corestructure containing the HP residue is relatively resistant to furtherproteolysis and provides a quantitative measure of the amount of type IIcollagen degraded.

Collagen type II is present in hyaline cartilage of joints in the adultskeleton. Quantitation of the collagen type II telopeptides in a bodyfluid, for example by way of a monoclonal antibody that recognizes anepitope in the peptide structure, would provide a quantitative measureof whole-body cartilage destruction or remodeling. In a preferredembodiment, the present invention involves an assay for cartilage tissuedegradation in humans based on quantifying the urinary excretion rate ofat least one member of this family of telopeptides. Such an assay couldbe used, for example, to:

(1) screen adult human subjects for those individuals having abnormallyhigh rates of cartilage destruction as an early diagnostic indicator ofosteoarthritis;

(2) monitor the effects of potential antiarthritic drugs on cartilagemetabolism in osteoarthritic and rheumatoid arthritic patients; or

(3) monitor the progress of degenerative joint disease in patients withosteoarthritis and rheumatoid arthritis and their responses to varioustherapeutic interventions.

Osteoarthritis is a degenerative disease of the articulating cartilagesof joints. In its early stages it is largely non-inflammatory (i.e.distinct from rheumatoid arthritis). It is not a single disease butrepresents the later stages of joint failure that may result fromvarious factors (e.g. genetic predisposition, mechanical overusage,joint malformation or a prior injury, etc.). Destruction of jointarticular cartilage is the central progressive feature ofosteoarthritis. The incidence of osteoarthritis, based on radiographicsurveys, ranges from 4% in the 18-24 year age group to 85% in the 75-79year age group. At present the disease can only be diagnosed by pain andradiographic or other imaging signs of advanced cartilage erosion.

The assays disclosed above may be used to detect early evidence ofaccelerated cartilage degradation in mildly symptomatic patients, tomonitor disease progress in more advanced patients, and as a means ofmonitoring the effects of drugs or other therapies.

In normal young adults (with mature skeletons) there is probably verylittle degradation of cartilage collagen. A test that could measurefragments of cartilage collagen in the urine (and in the blood and jointfluid) would be very useful for judging the “health” of cartilage in thewhole body and in individual joints. The type II collagen-specificpeptide assays described above will accomplish this. In the long term,such an assay could become a routine diagnostic screen for spottingthose individuals whose joints are wearing away. They could be targetedearly on for preventative therapy, for example, by the next generationof so-called chondroprotective drugs now being evaluated by the majorpharmaceutical companies who are all actively seeking better agents totreat osteoarthritis.

Other diseases in which joint cartilage is destroyed include: rheumatoidarthritis, juvenile rheumatoid arthritis, ankylosing spondylitis,psoriatic arthritis, Reiter's syndrome, relapsing polychondritis, thelow back pain syndrome, and other infectious forms of arthritis. Thetype II collagen-specific assays described herein could be used todiagnose and monitor these diseases and evaluate their response totherapy, as disclosed above in connection with osteoarthritis.

Type III Collagen Telopeptides

As pointed out above, human type III collagen telopeptides that may bepresent in body fluids are expected to have a core structure embodied inthe following parent structures:

is hydroxylysyl pyridinoline.

By analogy to the type II peptides, the type III collagen peptides mayoccur in glycosylated forms of the core structure. For example,galactose residues or glucosylgalactose residues may be attached to thecore structure, e.g. by way of hydroxyl groups.

The cross-linking residue of the type III collagen peptides is depictedas a 3-hydroxypyridinium residue, hydroxylysyl pyridinoline. The type IItelopeptide structures have been found to primarily have hydroxylysylpyridinoline cross-linking residues. However, whereas the type IIcollagen peptides are derived from the N-terminal telopeptide region oftype II collagen, the type III collagen peptides may be derived fromeither the N-terminal or the C-terminal of type III collagen, as long asat least one cross-linking residue is present.

Type III collagen is present in many connective tissues in associationwith type I collagen. It is especially concentrated in vascular walls,in the skin and in, for example, the synovial membranes of joints whereits accelerated turnover might be observed in inflammatory jointdiseases such as rheumatoid arthritis.

A specific assay for type III collagen degradation by quantitatingcross-linked type III collagen peptides as disclosed above, can be usedfor detecting, diagnosing, and monitoring various inflammatorydisorders, possibly with particular application to the vasculitissyndromes. In conjunction with assays for measuring bone type I andcartilage type II collagen degradation rates, such an assay could beused as a differential diagnostic tool for patients with variousdegenerative and inflammatory disorders that result in connective tissuedestruction or pathological processes.

Isolation of Type II and Type III Collagen Telopeptides

General Procedure:

Urine is collected form a normal adolescent during a rapid phase ofskeletal growth. Using a sequence of chromatographic steps that includebut are not limited to, adsorption on selective cartridges of ahydrophobic interaction support and an ion-exchange support andmolecular sieve, ion-exchange and reverse-phase HPLC columnchromatography steps, individual peptides are isolated. The cross-linkedpeptides containing HP (and LP) residues are detected during columnchromatography by their natural fluorescence (Ex max 297 nm<pH 4, Ex max330 nm, >pH 6; Em max 390 nm). An exemplary isolation procedure isprovided in the Example below.

Specific Example:

Fresh urine (at 4° C.) diluted 5 times with water and adjusted to 2%(v/v) trifluoroacetic acid, passed through a C-18 hydrophobic bindingcartridge (Waters C-18 Sep-pak prewetted with 80% (v/v) acetonitrilethen washed with water). Retained peptides were washed with water theneluted with 3 ml of 20% (v/v) acetonitrile, and this eluent was adjustedto 0.05 M NH₄HCO₃, 10% (v/v) acetonitrile by addition of an equal volumeof 0.1 M NH₄HCO₃. This solution was passed through a QMA-Sep-pak(Waters), which was washed with 10 ml of 0.1M NaCl, 20% (v/v)acetonitrile followed by 10 ml of water and the peptides were theneluted with 3 ml of 1% (v/v) trifluoroacetic acid and dried by Speed-Vac(Savant).

Peptides were fractionated in three chromatographic steps. The firststep was molecular sieve chromatography on a column of Bio-Gel P-10 (BioRad Labs, 2.5 cm×90 cm) eluted by 10% (v/v) acetic acid, monitoring theeffluent for HP fluorescence as shown in FIG. 2. In FIG. 2, the Y-axisis the relative fluorescence emission at 390 nm (297 nm excitation), andthe X-axis is the fraction number. The fraction size was 4 ml. Thefractions indicated as II are enriched in the cross-linked collagen typeII telopeptides. The cross-linked collagen type I telopeptides arecontained in the fractions indicated as III and IV. Fractions spanningpool II (enriched in the type II collagen cross-linked peptides) werecombined, freeze-dried and fractionated by ion-exchange columnchromatography on a DEAE-HPLC column (TSK-DEAE-5PW, 7.5 mm×7.5 mm,Bio-Rad Labs), equilibrated with 0.02 M Tris/HCl, 10% (v/v)acetonitrile, pH 7.5 and eluted with a gradient of 0-0.5M NaCl in thesame buffer as shown in FIG. 2.

FIG. 3A plots relative fluorescence emission at 390 nm (330 nmexcitation) versus elution time. The cross-linked collagen type IItelopeptides are found primarily in the segment indicated as IV. FIG. 3Bplots absorbance at 220 nm as a function of elution time in minutes.Pool IV contains the type II collagen cross-linked peptides. Individualpeptides were then resolved from pool IV by reverse phase HPLC on a C-18column (Aquapore RP-300, 25 cm×4.6 mm, Brownlee Labs), eluting with agradient of 0-30% (v/v) acetonitrile in 0.1% (v/v) trifluoroacetic acid.FIG. 4A shows a plot of relative fluorescence intensity at 390 nm (297nm excitation) as a function of elution time. The peaks associated withparticular peptides are indicated in FIG. 4A. FIG. 4B shows the relativeabsorbance at 220 nm as a function of time.

Cross-linked peptide fragments of type III collagen containing HPcross-linking residues may be isolated by a similar combination of stepsfrom the urine of normal growing subjects or, for example, from theurine of patients with inflammatory disorders of the vasculature.

Type I Collagen Telopeptides

This aspect of the invention is based on the discovery that both lysylpyridinoline (LP) and hydroxylysyl pyridinoline (HP) peptide fragments(i.e., telopeptides, as used herein) derived from reabsorbed bonecollagen are excreted in the urine without being metabolized. Theinvention is also based on the discovery that no other connectivetissues contain significant levels of LP and that the ratio of HP to LPin mature bone collagen remains relatively constant over a person'slifetime.

FIG. 5 compares the concentration of HP and LP in both cortical andcancellous human bone with age. It is observed that the concentration ofHP plus LP cross-links in bone collagen reaches a maximum by age 10 to15 years and remains reasonably constant throughout adult life.Furthermore, the ratio of HP to LP, shown in FIG. 6, shows little changethroughout life, remaining constant at about 3.5 to 1. These baselinedata demonstrate that the 3-hydroxypyridinium cross-links in bonecollagen remains relatively constant and therefore that body fluidsderived from bone collagen degradation will contain 3-hydroxypyridiniumcross-linked peptide fragments at concentrations proportional to theabsolute rate of bone resorption.

Since LP is the 3-hydroxypyridinium cross-link unique to bone collagen,the method for determining the absolute rate of bone resorption, in itssimplest form, is based on quantitating the concentration of peptidefragments containing 3-hydroxypyridinium cross-links and preferablylysyl pyridinoline (LP) cross-links in a body fluid.

As used in this description and in the appended claims with respect totype I, II, or III telopeptides, by “quantitating” is meant measuring byany suitable means, including but not limited to spectrophotometric,gravimetric, volumetric, coulometric, immunometric, potentiometric, oramperometric means the concentration of peptide fragments containing3-hydroxypyridinium cross-links in an aliquot of a body fluid. Suitablebody fluids include urine, serum, and synovial fluid. The preferred bodyfluid is urine.

Since the concentration of urinary peptides will decrease as the volumeof urine increases, it is further preferred that when urine is the bodyfluid selected, the aliquot assayed be from a combined pool of urinecollected over a fixed period of time, for example, 24 hours. In thisway, the absolute rate of bone resorption or collagen degradation iscalculated for a 24 hour period. Alternatively, urinary peptides may bemeasured as a ratio relative to a marker substance found in urine suchas creatinine. In this way the urinary index of collagen degradation andbone resorption would remain independent of urine volume.

In one embodiment of the present invention, monoclonal or polyclonalanti-bodies are produced which are specific to the peptide fragmentscontaining lysyl pyridinoline cross-links found in a body fluid such asurine. Type I telopeptide fragments may be isolated from a body fluid ofany patient, however, it is preferred that these peptides are isolatedfrom patients with Paget's disease or from rapidly growing adolescents,due to their high concentration of type I peptide fragments. Type II andtype III telopeptides may be isolated from a body fluid of any patientbut may be more easily obtained from patients suffering from diseasesinvolving type II or type III collagen degradation or from rapidlygrowing adolescents.

Isolation of Type I Collagen Telopeptides

Urine from patients with active Paget's disease is dialyzed in reducedporosity dialysis tubing (<3,500 mol. wt. cut off Spectropore) at 4° C.for 48 h to remove bulk solutes. Under these conditions the peptides ofinterest are largely retained. The freeze-dried non-diffusate is theneluted (200 mg aliquots) from a column (90 cm×2.5 cm) of Bio-Gel P2(200-400 mesh) in 10% acetic acid at room temperature. A region ofeffluent that combines the cross-linked peptides is defined by measuringthe fluorescence of collected fractions at 297 nm excitation/395 nmemission, and this pool is freeze-dried. Further resolution of thismaterial is obtained on a column of Bio-Gel P-4 (200-400 mesh, 90 cm×2.5cm) eluted in 10% acetic acid.

Two contiguous fraction pools are defined by monitoring the fluorescenceof the eluant above. The earlier fraction is enriched in peptidefragments having two amino acid sequences that derive from theC-terminal telopeptide domain of the αI(I) chain of bone type I collagenlinked to a third sequence derived from the triple-helical body of bonetype I collagen. These three peptide sequences are cross-linked with3-hydroxypyridinium. The overlapping later fraction is enriched inpeptide fragments having an amino acid sequence that is derived from theN-terminal telopeptide domain of bone type I collagen linked through a3-hydroxypyridinium cross-links.

Individual peptides are then resolved from each of the two fractionsobtained above by ion-exchange HPLC on a TSK DEAE-5-PW column (Bio Rad7.5 cm×7.5 mm) eluting with a gradient of NaCl (0-0.2M) in 0.02MTris-HCl, pH 7.5 containing 10% (v/v) acetonitrile. The N-terminaltelopeptide-based and C-terminal telopeptide-based cross-linked peptideselute in a series of 3-4 peaks of fluorescence between 0.08M and 0.15MNaCl. The C-terminal telopeptide-based cross-linked peptides elute firstas a series of fluorescent peaks, and the major and minor N-terminaltelopeptide-based cross-linked peptides elute towards the end of thegradient as characteristic peaks. Each of these is collected,freeze-dried and chromatographed on a C-18 reverse phase HPLC column(Vydac 218TP54, 25 cm×4.6 mm) eluted with a gradient (0-10%) ofacetonitrile: n-propanol (3:1 v/v) in 0.01M trifluoroacetic acid. About100-500 μg of individual peptide fragments containing3-hydroxypyridinium cross-links can be isolated by this procedure from asingle 24 h collection of Paget's urine.

Amino acid compositions of the major isolated peptides confirmed purityand molecular sizes by the whole number stoichiometry of recovered aminoacids. N-terminal sequence analysis by Edman degradation confirmed thebasic core structures corresponding to the sequences of the knowncross-linking sites in type I collagen and from the matching amino acidcompositions. The N-terminal telopeptide sequence of the α2(I) chain wasblocked from sequencing analysis due presumably to the known cyclizationof the N-terminal glutamine to pyrrolidone carboxylic acid.

A typical elution profile of N-terminal telopeptides obtained by theabove procedure is shown in FIG. 7A. The major peptide fragment obtainedhas an amino acid composition: (Asx)₂(Glx)₂(Gly)₅Val-Tyr-Ser-Thr, whereAsx is the amino acid Asp or Asn and Glx is the amino acid Gln or Glu.The sequence of this peptide is represented by Formula III below.

The C-terminal telopeptide-based cross-linked peptides resolved byreverse phase HPLC as described above are shown in FIG. 7B. As can beseen from this figure, these peptides are further resolved into a seriesof C-terminal telopeptides each containing the 3-hydroxypyridiniumcross-links. The major peptide, shown in FIG. 7B, was analyzed asdescribed above and was found to have the amino acid composition:(Asp)₅(Glu)₄(Gly)₁₀(His)₂(Arg)₂(Hyp)₂(Ala)₅. The sequence of thispeptide is represented by formula IV below. It is believed that theother C-terminal telopeptide-based cross-linked peptides appearing asminor peaks in FIG. 7B represent additions and deletions of amino acidsto the structure shown in Formula IV. Any of the peptides containedwithin these minor peaks are suitable for use as immunogens as describedbelow.

represents the HP or LP cross-links and Gln represents glutamine orpyrrolidone carboxylic acid.

Equivalents of the peptides represented by the above structures, interms of their presence in a body fluid due to collagen degradation,include those cases where there is some variation in the peptidestructure. Examples of such variation include 1-3 amino acid additionsto the N and C termini as well as 1-3 terminal amino acid deletions. Forexample, a peptide corresponding to Formula III, but having a tyrosineresidue attached to the amino terminus of the N-terminal aspartateresidue has been detected in relatively minor quantities in human urine.Smaller peptide fragments of the molecule represented by Formula IVderived from bone resorption are especially evident in urine. These arefound in the minor peaks of the C-terminal telopeptide fraction seen inFIG. 7B and can be identified by amino acid composition and sequenceanalysis.

EXAMPLES OF PROCEDURES FOR QUANTITATING PEPTIDES

A. Immunological Procedure for Quantitating Peptides

Immunological binding partners capable of specifically binding topeptide fragments derived from bone collagen obtained from aphysiological fluid can be prepared by methods well known in the art.The preferred method for isolating these peptide fragments is describedabove. By immunological binding partners as used herein is meantantibodies and antibody fragments capable of binding to a telopeptide.

Both monoclonal and polyclonal antibodies specifically binding thepeptides disclosed herein and their equivalents are prepared by methodsknown in the art. For example, Campbell, A. M. Laboratory Techniques inBiochemistry and Molecular Biology, Vol. 13 (1986). Elsevier, hereinincorporated by reference. It is possible to produce antibodies to theabove peptides or their equivalents as isolated. However, because themolecular weights of these peptide fragments are generally less than5,000, it is preferred that the hapten be conjugated to a carriermolecule. Suitable carrier molecules include, but are not limited to,bovine serum albumin, ovalbumin, thyroglobulin, and keyhole limpethemocyanin (KLH). Preferred carriers are thyroglobulin and KLH.

It is well known in the art that the orientation of the hapten, as it isbound to the carrier protein, is of critical importance to thespecificity of the anti-serum. Furthermore, not all hapten-proteinconjugates are equally successful immunogens. The selection of aprotocol for binding the particular hapten to the carrier proteintherefore depends on the amino acid sequence of the urinary peptidefragments selected. For example, if the peptide represented by FormulaIII is selected, a preferred protocol involves coupling this hapten tokeyhole limpet hemocyanin (KLH), or other suitable carrier, withglutaraldehyde. An alternative protocol is to couple the peptides to KLHwith a carbodiimide. These protocols help to ensure that the preferredepitope (discussed below under the heading “Characteristics of aPreferred Epitope)” are presented to the primed vertebrate antibodyproducing cells (e.g., B lymphocytes).

Other peptides, depending on the source, may require different bindingprotocols. Accordingly, a number of binding agents may be suitablyemployed. These include, but are not limited to, carbodiimides,glutaraldehyde, mixed anhydrides, as well as both homobifunctional andheterobifunctional reagents (see for example the Pierce 1986-87 catalog,Pierce Chemical Co., Rockford, Ill.). Preferred binding agents includecarbodiimides and heterobifunctional reagents such asm-Maleimidobenzyl-N-hydroxysuccinimide ester (MBS).

Methods for binding the hapten to, the carrier molecule are known in theart. See for example, Chard, T., Laboratory Techniques in Biochemistryand Molecular Biology, Vol. 6 (1987) Partz Elsevier, N.Y., hereinincorporated by reference.

Either monoclonal or polyclonal antibodies to the hapten-carriermolecule immunogen can be produced. However, it is preferred thatmonoclonal antibodies (MAb) be prepared. For this reason it is preferredthat immunization be carried out in the mouse. Immunization protocolsfor the mouse usually include an adjuvant. Examples of suitableprotocols are described by Chard, T. (1987) vida supra. Spleen cellsfrom the immunized mouse are harvested and homogenized and thereafterfused with cancer cells in the presence of polyethylene glycol toproduce a fused cell hybrid which produces monoclonal antibodiesspecific to peptide fragments derived from collagen. Examples of suchpeptides are represented by the formulas given above. Suitable cancercells include myeloma, hepatoma, carcinoma, and sarcoma cells. Detaileddescriptions of this procedure, including screening protocols, protocolsfor growing selected hybrid cells and harvesting monoclonal antibodiesproduced by the selected hybrid cells are provided in Galfre, G. andMilstein, C., Meth. Enzymol., 73:1 (1981). A preferred preliminaryscreening protocol involves the use of peptide fragments derived frombone collagen resorption and containing 3-hydroxypyridinium cross-linksin a solid phase radioimmunoassay. A specific example describing apreferred monoclonal antibody is provided below.

The monoclonal antibodies or other immunological binding partners usedin connection with the present are preferably specific for a particulartype of collagen telopeptide. For example, assays for the type II ortype III collagen degradation telopeptides should preferably be able todistinguish between the type I, type II, and type III peptides. However,in some cases, such selectivity will not be necessary, for example, ifit is known that a patient is not suffering degradation of one type ofcollagen but is suspected of suffering degradation from the assayed typeof collagen. Because of the differences in amino acid sequences betweenthe type I, type II, and type III families of telopeptides,cross-reactivity should not occur to a significant degree. Indeed,hybridomas can be selected for during the screening of splenocyte fusionclones that produce monoclonal antibodies specific for the cross-linkedtelopeptide of interest (and lack affinity for those of the other twocollagen types). Based on the differences in sequence of the isolatedpeptide structures, such specificity is entirely feasible. Peptidefragments of the parent types I, II and III collagens, suitable for suchhybridoma screening, can be prepared from human bone, cartilage andother tissues and used to screen clones from mice immunizedappropriately with the individual cross-linked peptide antigens isolatedfrom body fluid.

Immunological binding partners, especially monoclonal antibodies,produced by the above procedures, or equivalent procedures, are employedin various immunometric assays to quantitate the concentration of thepeptides having 3-hydroxypyridinium cross-links described above. Theseimmunometric assays preferably comprise a monoclonal antibody orantibody fragment coupled to a detectable marker. Examples of suitabledetectable markers include but are not limited to: enzymes, coenzymes,enzyme inhibitors, chromophores, fluorophores, chemiluminescentmaterials, paramagnetic metals, spin labels, and radionuclides. Examplesof standard immunometric methods suitable for quantitating thetelopeptides include, but are not limited to, enzyme linkedimmunosorbent assay (ELISA) (Ingvall, E., Meth. Enzymol., 70 (1981)),radio-immunoassay (RIA), and “sandwich” immunoradiometric assay (IRMA).

In its simplest form, these immunometric methods can be used todetermine the absolute rate of bone resorption or collagen degradationby simply contacting a body fluid with the immunological binding partnerspecific to a collagen telopeptide having a 3-hydroxypyridiniumcross-link.

It is preferred that the immunometric assays described above beconducted directly on untreated body fluids (e.g. urine, blood, serum,or synovial fluid). Occasionally, however, contaminating substances mayinterfere with the assay necessitating partial purification of the bodyfluid. Partial purification procedures include, but are not limited to,cartridge adsorption and elution, molecular sieve chromatography,dialysis, ion exchange, alumina chromatography, hydroxyapatitechromatography and combinations thereof.

Test kits, suitable for use in accordance with the present invention,contain specific binding partners such as monoclonal antibodies preparedas described above, that specifically bind to peptide fragments derivedfrom collagen degradation found in a body fluid. It is preferred thatthe specific binding partners of this test kit be coupled to adetectable marker of the type described above. Test kits containing apanel of two or more specific binding partners, particularlyimmunological binding partners, are also contemplated. Eachimmunological binding partner in such a test kit will preferably notcross-react substantially with a telopeptide, derived from another typeof collagen. For example, an immunological binding partner that bindsspecifically with a type II collagen telopeptide should preferably notcross-react with either a type I or type III collagen telopeptide. Asmall degree (e.g., 5-10%) of cross-reactivity may be tolerable. Othertest kits may contain a first specific binding partner to acollagen-derived telopeptide having a cross-link containing a pyridiniumring (which may be OH-substituted), and a second specific bindingpartner to a telopeptide having the same structure as the firsttelopeptide except that the pyridinium ring has been cleaved, such asphotolytically.

(i) Monoclonal Antibody Production

The following is an example of preparation of a monoclonal antibodyagainst a peptide immunogen based on Formula III above.

A fraction enriched in the peptide of Formula III (indicative of bonecollagen degradation) was prepared from adolescent human urine usingreverse phase and molecular sieve chromatography. The peptide wasconjugated to keyhole limpet hemocyanin (KLH) with glutaraldehyde usingstandard procedures. Mice (Balb/c) were immunized subcutaneously withthis conjugate (50-70 μg), first in complete Freund's adjuvant, thenboosted (25 μg) at 3 weekly intervals in incomplete Freund's adjuvantintraperitoneally. After test bleeds had shown a high titer against theFormula III peptide (referred to herein as P1) conjugated to bovineserum albumin (BSA) using an ELISA format, selected mice were boostedwith a low dose (5 μg) of the immunogen in sterile PBS intravenously.Three days later, cells from the spleens of individual mice were fusedwith mouse myeloma cells using standard hybridoma technology. Thesupematants of hybridoma clones growing in individual wells of 96-wellplates were screened for reactive monoclonal antibodies, initially usinga crude P1 preparation conjugated to BSA. After formal cloning bylimiting dilution, the antibodies produced by individual hybridomas werecharacterized against a panel of screening antigens using ELISAanalysis. These antigens were the P1 (Formula III) and P2 (Formula VII)peptides conjugated to BSA. An inhibition assay was used in which P1conjugated to BSA was plated out in the plastic wells, and antibody waspre-incubated with a solution of the potential antigen. A secondaryantibody (goat anti-mouse IgG conjugated to horseradish peroxidase, HRP)was used for color development using an appropriate substrate. Adesirable monoclonal antibody with high binding affinity for the P1peptide was identified. When used as an ascites fluid preparation, theantibody worked in an inhibition assay with optimal color yield at 2million-fold dilution (which indicates a binding constant in the rangeof 10⁻⁹ to 10⁻¹¹ M⁻¹, most likely about 10⁻¹⁰ M⁻¹). In an ELISA format,the antibody was able to detect and measure P1 present in normal humanurine without any concentration or clean-up steps. The hybridoma thatproduces this preferred monoclonal antibody has been deposited at theAmerican Type Culture collection (ATCC), 12301 Parklawn Drive,Rockville, Md. 20852, under accession number HB 10611 on Nov. 20, 1990.This hybridoma is designated below as 1H11; the monoclonal antibody itproduces is designated below as MAb-1H11.

Sandwich assays were also shown to work using the P1-specific monoclonalantibody and a polyclonal antiserum raised in rabbits against conjugatedP1. Either P1-specific monoclonal antibodies, polyclonal antiserum,binding fragments thereof, or the like can be used to bind specificallyto P1 from urine, in a detectable manner using standard ELISA and otherimmunoassay protocols.

(ii) Characteristics of a Preferred Epitope

The epitope recognized by the antibody MAb-1H11 is embodied in thestructure of P1. The epitope is recognized in pure P1 and in certainlarger peptides that contained the P1 structure (e.g., P1 attached to atyrosine residue via the N-terminal aspartate residue of P1). Theepitope includes chemical features of both of the two telopeptidesequences embodied in the structure of peptide P1. Peptides synthesizedto match the human α1 (I) and α2 (I) N-telopeptide sequences, with theaddition of a C-terminal cysteine for coupling to bovine serum albumin(i.e., Tyr-Asp-Glu-Lys-Ser-Thr-Gly-Gly-Cys (SEQ ID NO:2) andGln-Tyr-Asp-Gly-Lys-Gly-Val-Gly-Cys (SEQ ID NO:1)), were not recognizedby MAb-1H11. This was shown by ELISA using the free peptides competingagainst plated-out P1 (see FIG. 9) or directly as binding partnersconjugated to BSA and plated out. Referring to FIG. 9, the absorbance atλ=450 nm of a detectable marker is plotted against the concentration offree P1 peptide. As the amount of free P1 increases, the amount ofdetectable marker bound to immobilized (plated-out) P1 diminishes. Incomparison, the α2_(I) and α_(I) N-telopeptides demonstrate little ifany significant competitive binding with MAb-1H11.

In addition, a larger form of P1 bearing a tyrosine residue on theN-terminal aspartic acid was recovered from urine by affinity binding toMAb-1H11, but in lower yield than P1. Other slightly larger peptidesbearing the P1 epitope were also recovered but in even smaller amounts.

The antibody was not selective for the nature of the cross-link in P1,i.e., whether hydroxylysyl pyridinoline (HP) or lysyl pyridinoline (LP).Both HP-containing and LP-containing forms were bound, apparently withequal affinity, judging by the analysis of peptides isolated from urineby an affinity column consisting of MAb-1H11 coupled to agarose.

The free cross-linking amino acids, HP and LP, either made by acidhydrolysis from bone collagen or as present naturally in urine were notrecognized by MAb-1H11. After photolytic opening of the 3-pyridinol ringin peptide P1 with UV light (long UV wavelengths), specific antibodybinding was also unaffected, presumably because the individual peptidesremained cross-linked to each other. The epitope recognized by MAb-1H11,therefore, is made up of at least a combination of chemical andconformational features embodied in the two telopeptide sequences shownboxed in FIG. 10, together with steric features imposed by the trivalentcross-linking amino acid that links them. The α2 (I) N-telopeptidesequence, Gln-Tyr-Asp-Gly-Lys (SEO ID NO:3), is a particularlysignificant part of the epitope.

The fact that the epitope recognized by MAb-1H11 does not depend on anintact pyridinium ring is an unexpected discovery. If ring-openingoccurs either in vivo or even in vitro under routine handlingconditions, as appears likely, then a quantitative assay of the subjectpeptide(s) having intact pyridinium rings will underestimate the amountof bone resorption. Preliminary observations indicate that degradationof pyridinium rings in the subject peptides appears to occurparticularly in urine and/or in urine samples, even if refrigerated.Accordingly, an assay based on the present disclosure is expected to becomparatively more accurate. Two embodiments are envisioned: a singlespecific binding partner is employed that recognizes both closed andopen-ringed embodiments of the targeted peptide(s); or two specificbinding partners are employed, which differentiate between the closed-and open-ringed epitopes, respectively.

Further experiments showed that the epitope resides in human bonecollagen but is exposed and bound by MAb-1H11 only afler extensiveproteolysis. Thus, peptides produced from decalcified human bonecollagen by bacterial collagenase were bound by MAb-1H11 and shown to bederived from the N-telopeptide to helix site shown in FIG. 10. One formcontained the hexapeptide Gln-Tyr-Asp-Gly-Lys (Seq ID No:3) (in place onthe non-telopeptide K arm in P1), which is clearly derived from al (I)residues 928-933. Another form embodied an equivalent but distincthexapeptide that was derived from the α2 (I) chain. Fragments of humanbone collagen solubilized by pepsin, CNBr, or trypsin were notrecognized by MAb-1H11, either in an ELISA format when used ascompetitive inhibitors or on a Western blot after SDA-polyacrylamideelectrophoresis, indicating that these solubilizing agents do notproduce the epitope recognized by MAb-1H11.

B. Electrochemical Procedure for Assaying for Peptides

An alternative procedure for assaying for the above-described peptidesconsists of measuring a physical property of the peptides having3-hydroxypyridinium cross-links. One such physical property relies uponelectrochemical detection. This method consists of injecting an aliquotof a body fluid, such as urine, into an electrochemical detector poisedat a redox potential suitable for detection of peptides containing the3-hydroxypyridinium ring. The 3-hydroxypyridinium ring, being a phenol,is subject to reversible oxidation and therefore the electrochemicaldetector (e.g., Model 5100A Coulochem sold by Esa 45 Wiggins Ave.,Bedford, Mass.) is a highly desirable instrument suitable forquantitating the concentration of the present peptides. Two basic formsof electrochemical detector are currently commercially available:amperometric (e.g., BioAnalytical Systems) and coulometric (ESA, Inc.,Bedford, Mass. 01730). Both are suitable for use in accordance with thepresent invention, however, the latter system is inherently moresensitive and therefore preferred since complete oxidation or reductionof the analyzed molecule in the column effluent is achieved. Inaddition, screening or guard electrodes can be placed “upstream” fromthe analytical electrode to selectively oxidize or reduce interferingsubstances thereby greatly improving selectivity. Essentially, thevoltage of the analytical electrode is tuned to the redox potential ofthe sample molecule, and one or more pretreatment cells are set todestroy interferents in the sample.

In a preferred assay method, a standard current/voltage curve isestablished for standard peptides containing lysyl pyridinoline orhydroxylysyl pyridinoline in order to determine the proper voltage toset for optimal sensitivity. This voltage is then modified dependingupon the body fluid, to minimize interference from contaminants andoptimize sensitivity. Electrochemical detectors, and the optimumconditions for their use are known to those skilled in the art. Complexmixtures of body fluids can often be directly analyzed with theelectrochemical detector without interference. Accordingly, for mostpatients no pretreatment of the body fluid is necessary. In some caseshowever, interfering compounds may reduce the reliability of themeasurements. In such cases, pretreatment of the body fluid (e.g.,urine) may be necessary.

Accordingly, in an alternative embodiment of the invention, a body fluidis first purified prior to electrochemically titrating the purifiedpeptide fragments. The purification step, may be conducted in a varietyof ways including but not limited to dialysis, ion exchangechromatography, alumina chromatography, hydroxyapatite chromatography,molecular sieve chromatography, or combinations thereof. In a preferredpurification protocol, a measured aliquot (25 ml) of a 24 hour urinesample is dialyzed in reduced porosity dialysis tubing to remove thebulk of contaminating fluorescent solutes. The non-diffusate is thenlyophilized, redissolved in 1% heptafluorobutyric acid (HFBA), an ionpairing solution, and the peptides adsorbed on a Waters Sep-Pak C-18cartridge. This cartridge is then washed with 5 ml of 1% HFBA, and theneluted with 3 ml of 50% methanol in 1% HFBA.

Another preferred method of purification consists of adsorbing ameasured aliquot of urine onto an ion-exchange adsorption filter andeluting the adsorption filter with a buffered eluting solution. Theeluate fractions containing peptide fragments having 3-hydroxypyridiniumcross-links are then collected to be assayed.

Still another preferred method of purification employs molecular sievechromatography. For example, an aliquot of urine is applied to a Bio-GelP2 or Sephadex G-20 column and the fraction eluting in the 1000-5000Dalton range is collected. It will be obvious to those-skilled in theart that a combination of the above methods may be used to purify orpartially purify urine or other body fluids in order to isolate thepeptide fragments having 3-hydroxypyridinium cross-links. The purifiedor partially purified peptide fragments obtained by the above proceduresmay be subjected to additional purification procedures, furtherprocessed or assayed directly in the partially purified state.Additional purification procedures include resolving partially purifiedpeptide fragments employing high performance liquid chromatography(HPLC) or microbore HPLC when increased sensitivity is desired. Thesepeptides may then be quantitated by electrochemical titration.

A preferred electrochemical titration protocol consists of tuning theredox potential of the detecting cell of the electrochemical detector(Coulochem Model 5100A) for maximum signal with pure HP. The detector isthen used to monitor the effluent from a C-18 HPLC column used toresolve the partially purified peptides.

C. Fluorometric Procedure for Quantitating Peptides

An alternative preferred method for quantitating the concentration ofpeptides having 3-hydroxypyridinium cross-links as described herein isto measure the characteristic natural fluorescence of these peptides.For those body fluids containing few naturally occurring fluorescentmaterials other than the 3-hydroxypyridinium cross-links, fluorometricassay may be conducted directly without further purification of the bodyfluid. In this case, the peptides are resolved by HPLC and the naturalfluorescence of the HP and LP amino acid residues is measured at 395 nmupon excitation at 297 nm, essentially as described by Eyre, D. R., etal., Analyte. Biochem. 137:380 (1984), herein incorporated by reference.

It is preferred, in accordance with the present invention, that thefluorometric assay be conducted on urine. Urine, however, usuallycontains substantial amounts of naturally occurring fluorescentcontaminants that must be removed prior to conducting the fluorometricassay. Accordingly, urine samples are first partially purified asdescribed above for electrochemical detection. This partially purifiedurine sample can then be fluorometrically assayed as described above.Alternatively, the HP and LP cross-linked peptides in the partiallypurified urine samples or other body fluids can be hydrolyzed in 6M HClat about 108° C. for approximately 24 hours as described by Eyre, et al.(1984) vida supra. This process hydrolyzes the amino acids connected tothe lysine precursors of “tripeptide” HP and LP cross-links, producingthe free HP and LP amino acids represented by Formulae I and II. Thesesmall “tripeptides” are then resolved by the techniques described above,preferably by HPLC, and the natural fluorescence is measured (Ex 297 nm,Ex 390 nm).

Optionally, the body fluid (preferably urine) is passed directly througha C-18 reverse phase affinity cartridge after addingacetonitrile/methanol 5 to 10% V/V. The non-retentate is adjusted to0.05-0.10M with a cationic ion-pairing agent such as tetrabutyl ammoniumhydroxide and passed through a second C-18 reverse phase cartridge. Thewashed retentate, containing fluorescent peptides, from this secondcartridge is eluted with acetonitrile:water (or methanol:water), driedand fluorescent peptides are analyzed by reverse phase HPLC or microboreHPLC using an anionic ion-pairing agent such as 0.01M trifluoroaceticacid in the eluant.

FIG. 8A displays the elution profile resolved by reverse phase HPLC ofnatural fluorescence for a hydrolysate of peptide fragments from normalhuman urine. Measurement of the integrated area within the envelope of agiven component is used to determine the concentration of that componentwithin the sample. The ratio of HP:LP found in normal human urine andurine from patients having Paget's disease, FIG. 8B, are bothapproximately 4.5:1. This is slightly higher than the 4:1 ratio found inbone itself (Eyre, et al., 1984). The higher ratio found in urineindicates that a portion of the HP fraction in urine may come fromsources other than bone, such as the diet, or other sources of collagendegradation, i.e., cartilage catabolism. It is for this reason that itis preferred that LP which derives only from bone be used to provide anabsolute index of bone resorption. However, in the absence of excessivecartilage degradation such as in rheumatoid arthritis or in cases wherebone is rapidly being absorbed, HP or a combination of HP plus LP may beused as an index of bone resorption.

While the invention has been described in conjunction with preferredembodiments, one of ordinary skill after reading the foregoingspecification will be able to effect various changes, substitutions ofequivalents, and alterations to the subject matter set forth herein.Hence, the invention can be practiced in ways other than thosespecifically described herein. It is therefore intended that theprotection granted by Letters Patent hereon be limited only by theappended claims and equivalents thereof.

1. A method of assaying type I collagen fragments in a body fluidsample, comprising: (a) contacting the body fluid with a syntheticpeptide consisting essentially of either the type I collagen α1(I)N-telopeptide sequence Asp-Glu-Lys-Ser-Thr-Gly-Gly (SEQ ID NO:5) or theα2(I) N-telopeptide sequence Gln-Tyr-Asp-Gly-Lys-Gly-Val-Gly (SEQ IDNO:6), and an immunological binding partner immunoreactive with saidamino acid sequence, wherein the collagen fragments compete with saidsynthetic peptide for binding with the immunological binding partner;and (b) quantifying the amount of collagen fragments in the sample bymeasuring the amount of binding of said immunological binding partnerwith said collagen fragments.
 2. The method of claim 1, wherein theimmunological binding partner is a monoclonal antibody or animmunologically active fragment thereof.
 3. The method of claim 1,wherein the body fluid is urine.
 4. The method of claim 1, wherein thesample is obtained from a post-menopausal woman.
 5. The method of claim1, wherein the amount of type I collagen fragments in the body fluidsample is quantified with reference to the amount of binding between theimmunological binding partner and one or more standard samples.
 6. Animmunological binding partner that binds to either the type I collagenα1(I) N-telopeptide sequence Asp-Glu-Lys-Ser-Thr-Gly-Gly (SEQ ID NO:5)or the α2(I) N-telopeptide sequence Gln-Tyr-Asp-Gly-Lys-Gly-Val-Gly (SEQID NO:6), wherein said immunological binding partner is raised against asynthetic peptide consisting essentially of said amino acid sequence. 7.The immunological binding partner of claim 6 that is a monoclonalantibody or immunologically active fragment thereof.
 8. A cell linewhich produces a monoclonal antibody that binds to either the type Icollagen α1(I) N-telopeptide sequence Asp-Glu-Lys-Ser-Thr-Gly-Gly (SEQID NG:5) or the α2(I) N-telopeptide sequenceGln-Tyr-Asp-Gly-Lys-Gly-Val-Gly (SEQ ID NO:6), wherein saidimmunological binding partner is raised against a synthetic peptideconsisting essentially of said sequence.
 9. A test kit for assaying typeI collagen fragments in a body fluid sample, comprising theimmunological binding partner of claim
 6. 10. A test kit for assayingtype I collagen fragments in a body fluid sample, comprising themonoclonat antibody of claim 7.